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BACKGROUND: Skin transplantation is one of the most effective methods for treating large-area burns. How to effectively suppress the immune rejection after allogeneic skin transplantation is a problem that needs to be solved urgently. OBJECTIVE: To investigate the effect of human adipose derived mesenchymal stem cells (hADSCs) on the immunoregulation of skin grafts in different strains of mice. METHODS: Isolated hADSCs were cultured to the 3rd generation. Sixty ICR neonatal mice, 2-4 days of age, were randomly divided into four groups (n=15). The skin tissues of ICR neonatal mice were transplanted into adult C57BL/6 mice to establish a different strain of mouse skin graft immune rejection model. PBS and low dose (5×104), medium dose (10×104), high dose (20×104) hADSCs were injected into the model mice through tail vein, and the survival time of transplanted skin in each group was recorded. On the 7th day after operation, five mice from each group were randomly selected to remove their spleen and serum, and the expression of immune factors interleukin-10, tumor necrosis factor-α and interferon-γ were detected by RT-PCR and ELISA respectively. The transplanted part of the skin was taken to make pathological sections for observing the infiltration of lymphocytes. RESULTS AND CONCLUSION: Compared with the PBS group, the survival time of the skin was prolonged in the low dose hADSCs group; however, there was no significant difference between the two groups (P > 0.05). Compared with the PBS and low dose hADSCs groups, the survival time of the skin was significantly increased in the medium and high dose groups (P 0.05). Compared with the PBS group, the relative expression of tumor necrosis factor-α and interferon-γ in the spleen and serum was significantly decreased in the low, medium and high dose hADSCs groups (P < 0.05), whereas the level of interleukin-10 was significantly elevated in the medium and high dose hADSCs groups (P < 0.05). To conclude, the appropriate dose of hADSCs can significantly prolong the survival time of transplanted skin between different strains of mice, by regulating the expression of related immune factors in the recipient mice.
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Objective:To compare the ability between bone marrow-derived mesenchymal stem cells (MSCs) (BM-MSCs) and adipose-derived MSCs (AD-MSCs) or umbilical cord-derived MSCs (UC-MSCs) on promotion of vessels formation and vessds stabilization relevant to the functions of EPCs.Methods:In vitro,co-culture blood vessel test was performed to compare the angiogenic ability between BM-MSCs,AD-MSCs or UC-MSCs.In vivo,angiogenic assay dependent on basement membrane matrix Matrigel and immunohistochemistry were performed to compare the ability of vessels formation functions between BM-MSCs and AD-MSCs or UC-MSCs.Results:The lengths and dots of vascular structures formed by EPCs on AD-MSCs layer are greater than those by EPCs on BM-MSCs layer and UC-MSCs layer in angiogenic assay in vitro.The stability of the capillary-like structures formed by EPCs with AD-MSCs on Matrigel was more stable than that by the BM-MSCs,UC-MSCs or EPCs.AD-MSCs and EPCs could form abundant functional vessels with blood perfusion in Matrigelin vivo;UC-MSCs and EPCs could form a few functional vessels with blood perfusion in Matrigelin vivo;BM-MSCs and EPCs could form broken vessels with hemocytes leakage in Matrigel in vivo.Conclusion:AD-MSCs have the stronger ability to promote the angiogenesis and stabilize the vessels compared with BM-MSCs or UC-MSCs ex vivo and in vivo.
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Objective To investigate the effects of ZEB1 (Zinc finger E-box binding homeobox 1) gene knockdown on proliferation, cell cycle and apoptosis of human adipose-derived mesenchymal stem cells (hADSCs).Methods Primary mesenchymal stem cells were obtained from human adipose tissue by collagenase digestion and identified by immunological phenotype and differentiation potential of osteogenic and adipogenic lineages .The siRNA was trans-fected into hADSCs by lipofectamine 2000 .The expression of ZEB 1 and key proliferation , cell cycle and apoptosis-related genes were detected by real-time PCR and Western blot at mRNA and protein level respectively .The cell proliferation capacity was assessed by MTS .The apoptosis and cell cycle of hADSCs were evaluated by flow cytome-try.Results The ZEB1 expression at mRNA and protein level was significantly decreased in si-ZEB1 transfection cells as compared to transfection with si-NC.Inhibition of ZEB1 decreased the cell proliferation ability , and signifi-cantly inhibited the expression of cell proliferation related genes, including CCND1, MKI67, MYC and PCNA.After transfection with si-ZEB1 , the percentage of cells in G1 phase increased from 50.17% to 58.94%, and S phase and G2 phase decreased by 6.16% and 2.07% separately.Meanwhile, the apoptosis rate increased by 10.2%, the expression of apoptosis related genes TP53 and BAX was up-regulated , and the expression of anti-ap-optotic genes MCL1 and BCL2 was down-regulated in si-ZEB1 transfection cells as compared to transfection with si-NC.Conclusions ZEB1 may play an important role in promoting hADSCs proliferation , cell cycle progression and inhibit their apoptosis .
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BACKGROUND AND OBJECTIVES: Adipose-derived mesenchymal stem cells (ADSCs) are promising candidates in regenerative medicine. The need for in vitro propagation to obtain therapeutic quantities of the cells imposes a risk of impaired functionality due to cellular senescence. The aim of the study was to analyze in vitro senescence of previously cryopreserved human ADSCs subjected to serial passages in cell culture. METHODS AND RESULTS: ADSC cultures from 8 donors were cultivated until proliferation arrest was reached. A gradual decline of ADSC fitness was observed by altered cell morphology, loss of proliferative, clonogenic and differentiation abilities and increased β-galactosidase expression all of which occurred in a donor-specific manner. Relative telomere length (RTL) analysis revealed that only three tested cultures encountered replicative senescence. The presence of two ADSC subsets with significantly different RTL and cell size was discovered. The heterogeneity of ADSC cultures was supported by the intermittent nature of aging seen in tested samples. CONCLUSION: We conclude that the onset of in vitro senescence of ADSCs is a strongly donor-specific process which is complicated by the intricate dynamics of cell subsets present in ADSC population. This complexity needs to be carefully considered when elaborating protocols for personalized cellular therapy.
Subject(s)
Humans , Aging , Cellular Senescence , Cell Culture Techniques , Cell Size , Mesenchymal Stem Cells , Population Characteristics , Regenerative Medicine , Serial Passage , Telomere , Tissue DonorsABSTRACT
Objective To investigate the role of human adipose-derived mesenchymal stem cells (ADSC) cultured with astragaloside Ⅳ(Ast) on renal tubular epithelial cells line (HKC) induced by cisplatin in vitro.Methods HKC was divided into four groups:(1) normal control group; (2)cisplatin group; (3) ADSC and HKC group; (4) ADSC cultured with Ast and HKC group.After coincubated with ADSC,the apoptosis index and proliferation of HKC were detected respectively.The levels of interleukin-6 (IL-6),regulated upon activation normal T cell expressed and secreted (RANTES),erythropoietin (EPO) and insulin like growth factor 1 (IGF-1) were detected by ELISA.Transwell culture system were used to test the migration effect of ADSC.Results Compared with cisplatin group,the apoptotic rates of HKC were decreased and the cell number increased obviously (P ＜ 0.05) in ADSC and HKC group and ADSC cultured with Ast and HKC group.Compared with cisplatin group,the expression of IL-6 and RANTES were lower (P ＜ 0.05),and the level of EPO and IGF-1 increased significantly (P ＜ 0.05),in ADSC cultured with Ast and HKC group.Crystal violet staining showed that ADSC cultured with Ast and HKC group migration index was higher than the other three groups (P ＜ 0.05).Conclusion The intervention of Ast may stimulate paracrine effect and migration of ADSC,so as to reduce apoptosis of HKC.